https://scholars.lib.ntu.edu.tw/handle/123456789/160372
DC 欄位 | 值 | 語言 |
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dc.contributor | 李建國 | zh_TW |
dc.contributor | 臺灣大學:免疫學研究所 | zh_TW |
dc.contributor.author | 劉大鳴 | zh |
dc.contributor.author | Liu, Ta-Ming | en |
dc.creator | 劉大鳴 | zh |
dc.creator | Liu, Ta-Ming | en |
dc.date | 2005 | en |
dc.date.accessioned | 2007-11-29T04:56:41Z | - |
dc.date.accessioned | 2018-07-09T01:37:07Z | - |
dc.date.available | 2007-11-29T04:56:41Z | - |
dc.date.available | 2018-07-09T01:37:07Z | - |
dc.date.issued | 2005 | - |
dc.identifier | en-US | en |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/63309 | - |
dc.description.abstract | B淋巴球的發育初始於幹細胞,並且在骨髓中分化完全,這個過程受到相當嚴密的調控。成熟的B淋巴球會轉移至脾臟中,當受到外來抗原的刺激後,將會進一步分化成為漿細胞並產生特定抗體。B淋巴球複雜的發育過程受到許多細胞激素與基因調控蛋白的作用。STAT1是STAT家族中的一員,這類調控蛋白是透過傳遞上游細胞激素的訊息,進一步調節下游對應基因的表現。目前對於STAT1在B細胞發育上所扮演的角色仍然不清楚。在本論文中,我們發現STAT1基因剔除小鼠的早期B淋巴球發育,不論在pro-B, pre-B, immature-B, 或mature-B時期皆有明顯的缺陷;特別的是,STAT1基因剔除小鼠的骨髓細胞(B220+CD138+)則有明顯減少的現象。 從體外分化實驗的研究中發現,經由LPS刺激活化的STAT1基因剔除脾臟細胞有較少的抗體產生;並且在漿細胞分化調控基因上有不正常的表現量。相反的是,當這些STAT1基因剔除B淋巴球從脾臟細胞純化出來並接受LPS刺激活化後,抗體的生成與漿細胞的分化則無明顯差異,這似乎暗示前者的現象可能是受到B淋巴球以外的細胞調控。有趣的是, IL-10在刺激活化後的分泌量明顯減少。 為了進一步探討STAT1對於B淋巴球發育上的角色,我們利用T-dependent與T-independent兩種不同抗原來免疫STAT1基因剔除小鼠與正常小鼠。研究中發現,免疫後的STAT1基因剔除小鼠的骨髓與脾臟CD138+的細胞有明顯減少的現象。經由T-independent 抗原免疫後的STAT1基因剔除小鼠血清中,IgM的生成量較少; 另一方面,經由T-dependent 抗原免疫後的STAT1基因剔除小鼠血清中,IgG的生成量則明顯的減少。進一步我們也發現,經由TD抗原免疫後的STAT1基因剔除小鼠血清中的IgG1,IgG2a,IgG2b皆明顯減少,這暗示著在STAT1基因剔除小鼠中產生抗體的缺陷並非完全導因於IFN-γ的不正常反應。 綜合以上的結果,STAT1對於早期B淋巴球的發育及漿細胞的分化都扮演著重要的角色。更進一步驗證了STAT1基因在體液免疫反應的意外的角色。 | zh_TW |
dc.description.abstract | B cell development is a tightly regulated process which originates from hematopoiesis stem cells (HSCs) and commits to B-lineage in BM. Mature B cells migrate to spleen and terminally differentiate into antibody-secreting plasma cells after encountering foreign antigens. The complicated B cell developmental progresses are orchestrated by many cytokines and transcriptional cascades. STAT1 is a member of the STAT family which transmits cytokine signals and induces down-stream gene expression. The role of STAT1 in B cell development is still largely unknown. Using ST1KO mice, we have demonstrated that early B lymphopoiesis is impaired in the absence of STAT1, including reduced numbers of pro-B, pre-B, immature-B, and mature-B cells in BM. Interestingly, B220+CD138+ cells of ST1KO mice are also significantly decreased in BM as apposed to WT mice. In vitro differentiation indicates that reduced Ig production and dysregulation of genes that are critical for plasma cell formation are observed in ST1KO splenocytes upon LPS stimulation. By contrast, comparable Ig production and normal plasma cell differentiation are observed in purified B cell of ST1KO mice after LPS stimulation, implying that the effects seen in splenocytes is through non-B-cell-dependent mechanisms. Interestingly, levels of IL-10 are significantly reduced after LPS stimulation. To further investigate the role of STAT1 in B cell differentiation, ST1KO mice are immunized with T-dependent (TD) or T-independent (TI) antigens in vivo. Surprisingly, plasma cells of ST1KO mice are dramatically reduced in BM or SP after immunization. Correspondingly, reduced antigen-specific IgM production is observed in response to a TI antigen, and impaired total IgG but not IgM is observed in response to TD antigen. In addition, the failure of class switching into IgG1, IgG2a and IgG2b in ST1KO mice in response to TD antigen, suggesting that this effect is not entirely due to the loss of IFN-γ-responsiveness. In summary, ST1KO mice reveal defects in early B lymphopoiesis and impaired plasma cell differentiation. This further confirms the unexpected role of STAT1 in humoral immune responses. | en |
dc.description.tableofcontents | 誌謝 i Abbreviations ii 摘要 iv Abstract vi Table of Contents viii Chapter 1 Introduction 1 1.1 JAK-STAT pathway 1 1.2 STAT1 2 1.3 B cell development 3 1.4 B cell differentiation 3 1.5 Rationale 4 1.6 Specific aims 6 Chapter 2 Materials and Methods 8 2.1 Mice 8 2.2 Flow Cytometry 8 2.3 Cell Culture 9 2.4 Purification of B cells 9 2.5 Calcium phosphate-mediated transfection 9 2.6 Western Blotting 10 2.7 BrdU Incorporation Assay 10 2.8 Preparation of cDNA using RNAtureTM kit 10 2.9 Quantitative Real-Time PCR 11 2.10 ELISA 12 2.11 CBA assay 13 2.12 3H-Thymidine incorporation assay 13 2.13 CFSE labeling 13 2.14 In vivo immunization 14 2.15 Construction of pTAP-STAT1s 14 Chapter 3 Results 15 3.1 B cell development is impaired in ST1KO mice. 15 3.2 Reduced humoral response and plasma cell differentiation in ST1KO B cells upon LPS stimulation in vitro. 16 3.3 Comparable plasma cell differentiation and Ig secretion in vitro in purified B cells from WT and ST1KO mice. 20 3.4 Impaired plasma cell formation and IgM production in ST1KO mice in response to T-independent antigen in vivo. 24 3.5 Impaired plasma cell formation and IgG production in ST1KO mice in response to T-dependent antigen in vivo. 25 3.6 Construction of expression plasmids that express tagged-STAT1s for TAP system. 26 3.7 Expression and functional assay for pTAP-STAT1s. 28 Chapter 4 Discussion 30 4.1 Impaired B lymphopoiesis in ST1KO mice. 30 4.2 Impaired in vitro plasma cell formation and Ig secretion in ST1KO splenic B cells but not in purified ST1KO B cells. 31 4.3 Impaired plasma cell formation and Ig production in ST1KO mice in vivo. 34 Reference 37 Figures and Tables 42 Table 1 Cellularity of bone marrow is slightly decreased in STAT1 knockout mice. 43 Fig. 1 STAT1 is completely deleted at mRNA and protein level of ST1KO spleen.. 44 Fig. 2 Different B cell lineages are decreased in the bone marrow of ST1KO mice. 45 Fig. 3 Slight decrease of percentage of B lymphocytes in spleen and peripheral blood of ST1KO mice. 46 Fig. 4 Bone marrow B220+CD138+ cells are significantly decreased in ST1KO mice. 47 Fig. 5 STAT1 is activated in splenic lymphocytes upon LPS stimulation.. 48 Fig. 6 Comparable proliferation of WT and ST1KO lymphocytes in response to LPS. 49 Fig. 7 Comparable plasma cell differentiation of WT and ST1KO splenocytes in vitro. 50 Fig. 8 Production and mRNA expression of sIgM is reduced in ST1KO cells upon LPS stimulation. 51 Fig. 9 Dysregulation of genes that are critical for plasma cell differentiation in ST1KO cells upon LPS stimulation. 52 Fig. 10 IL-10 secretion is reduced in ST1KO splenic cells upon LPS stimulation. 53 Fig. 11 Decreased proliferation of purified ST1KO B cells upon LPS stimulation. 54 Fig. 12 Comparable percentage of CD138+b220+ cell in purified splenic B cells of WT and ST1KO mice. 55 Fig. 13 CD138+ B cells of ST1KO mice divide slowly in the earlier stage after LPS treatment.. 56 Fig. 14 Slightly decreased production sIgM by purified B cells of ST1KO mice. 57 Fig. 15 Comparable levels of genes that are critical for plasma cell differentiation in the purified B cells of WT and ST1KO mice. 58 Fig. 16 Similar levels of IL-10 and IL-4 mRNA in WT and ST1KO splenic B cells upon LPS stimulation. 59 Fig. 17 Decreased percentage and number of CD138+ cells in bone marrow and spleen of ST1KO mice and reduced production of TNP-specific sIgM in vivo in response to T-independent antigens. 61 Fig. 18 Decreased percentage of CD138+ cells in bone marrow and spleen of ST1KO mice and reduced production of TNP-specific IgG in vivo in response to T-dependent antigen. 63 Fig. 19 Reduced production of TNP-specific IgG subclass in vivo in response to T-dependent antigen. 64 Fig 20 Schematic diagram of pNTAP-STAT1 constructs. 65 Fig 21 Schematic diagram of pCTAP-STAT1 constructs. 66 Fig. 22 High transfection efficiency of GFP-reporting construct in 293T cells.. 67 Fig. 23 WT or mutant pNTAP-STAT1 and pCTAP-STAT1 constructs are expressed in 293T cells.. 68 Fig. 24 Functional assay for mutated STAT1 constructs using co-transfection of JAK1.. 69 Fig. 25 Functional assay for mutated STAT1 constructs using human IFN-α stimulation.. 70 | en |
dc.format.extent | 1061812 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | en-US | en |
dc.language.iso | en_US | - |
dc.subject | 淋巴球 | en |
dc.subject | STAT1 | en |
dc.title | STAT1在B淋巴球發育及分化上所扮演角色之研究 | en |
dc.title | The Role of STAT1 in B cell Development and differentiation | en |
dc.type | other | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/63309/1/ntu-94-R92449007-1.pdf | - |
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Induction of IgG2a class switching in B cells by IL-27. J Immunol 173, 2479-2485. | en |
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item.openairetype | other | - |
item.languageiso639-1 | en_US | - |
item.grantfulltext | open | - |
item.cerifentitytype | Products | - |
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